Method of preparing pharmaceutical compositions

ABSTRACT

A method for the discovery of compounds suitable for the treatment and/or prophylaxis of obesity, in which the ability of the test compounds to inhibit de novo lipogenesis in mammals and/or man is determined. The use of compounds which are capable of inhibiting de novo lipogenesis in mammals, and which are substantially free of effects directed towards the CNS, for the preparation of pharmaceutical compositions for the treatment and/or prophylaxis of obesity, as well as for the treatment and/or inhibition of obesity, are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/907,440,filed Jul. 18, 2001, now U.S. Pat. No. 6,946,243, issued Sep. 20, 2005,which is an application claiming the benefit under 35 U.S.C. §119(e) ofU.S. provisional application Ser. No. 60/219,672, filed Jul. 21, 2000,which claims the priority to German patent Application No. 100 35 227.8,filed Jul. 20, 2000, the entire disclosures of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates to a method of identifying compoundssuitable for the treatment and/or prophylaxis of obesity. The inventionfurther relates to the use of compounds which are capable of inhibitingde novo lipogenesis in mammals, and which are substantially free ofeffects directed towards the central nervous system (=CNS), for thepreparation of drugs for the treatment and/or prophylaxis of obesity.

Today, especially in the developed industrial nations, obesity is anincreasingly serious problem for the health of the population, beingcaused predominantly by unbalanced and excessively high-fat nutrition.The increase in the percentage of overweight people in the population isbeing accompanied by an increase in the consequences of obesity, whichrange from personal discontentment to cardiovascular disease or certainforms of diabetes. There are therefore already a number of therapeuticprocedures aimed at the treatment or prophylaxis of obesity. One examplewhich may be mentioned is lipase-inhibitory compounds, which reducelipolysis in the intestinal tract and thereby cut down the energy yieldfrom the food intake. Thus, in this therapeutic procedure, at least partof the alimentary fats is excreted undecomposed. It is however desirableto have other novel therapeutic procedures for the treatment and/orprophylaxis of obesity which can complement the previously known formsof therapy.

SUMMARY OF THE INVENTION

It has now been found, surprisingly, that compounds which are capable ofinhibiting de novo lipogenesis in mammals, especially man, areadvantageously suitable for the effective treatment and/or prophylaxisof obesity. Particularly good results are achieved by administering theabove-mentioned compounds over prolonged periods, for example forperiods of several weeks.

The invention thus relates to a method of discovering compounds suitablefor the treatment and/or prophylaxis of obesity, in which thosecompounds are selected which are capable of inhibiting de novolipogenesis in mammals, especially man. Furthermore, the invention alsorelates to the use of compounds which are capable of inhibiting de novolipogenesis in mammals, and which are in particular substantially freeof effects directed towards the CNS, such as anticonvulsant effects, forthe preparation of pharmaceutical compositions for the treatment and/orprophylaxis of obesity, as well as to a method of treating or inhibitingobesity.

De novo lipogenesis (abbreviated hereafter to DNL) is understood asmeaning the synthesis of endogenous fatty acids from carbohydrates inthe mammalian organism. This synthetic reaction takes place in thecytosol of body cells and is based on the so-called citric acid cycle orKrebs-Martius cycle. In this cycle, in an endogenous biochemicalreaction, citrate is ultimately synthesized from the two componentspyruvate, which originates from carbohydrates, and bicarbonate viadifferent intermediates—including maleate, fumarate and α-ketoglutarate.If citrate is synthesized in excess, it can be converted via theintermediate acetyl-coenzyme A to free fatty acids (=lipidic subsequentproducts), which form fats and can then be stored in fat cells(=adipocytes). The excessive storage of fats formed from fatty acids inbody cells can quite generally lead to obesity. A variety of enzymesparticipate in the citric acid cycle. The turnover of the citrate cycledepends substantially on the amount of bicarbonate available. The amountof bicarbonate available in turn depends on the rate at which it can beformed from carbon dioxide. This bicarbonate-yielding equilibriumreaction is catalyzed by so-called carboanhydrases. Of the knowncarboanhydrases and isozymes thereof, predominantly carboanhydraseisozymes of subtypes II (=CA II) and V (=CA V) participate, in mammals,in the catalysis of reactions which provide bicarbonate for the citricacid cycle. Carboanhydrases of subtype V, which are present in themitochondria, have a particular role to play. The citric acid cycle alsotakes place in the mitochondria.

There are various conceivable possibilities for inhibiting DNL inmammalian cells, all of which aim to reduce the turnover of the citricacid cycle. This makes it possible to cut down the concentration ofcitrate produced in excess, which is available for the synthesis offatty acids. According to the present invention, DNL can preferably beinhibited by inhibiting the carboanhydrases which catalyze the reactionsyielding bicarbonate for the citric acid cycle. For the purpose of theinvention, it is possible to inhibit preferably carboanhydrases ofsubtypes II and/or V and particularly preferably CA V.

Compounds which are capable of inhibiting carboanhydrasesnon-specifically (=non-specific or conventional CA inhibitors) are knownper se and have been used for a relatively long time in varioustherapeutic fields, mainly as diuretics or in ophthalmology. A survey offields of use of such conventional CA inhibitors can be found e.g. in C.T. Supuran, Expert Opinion on Therapeutic Patents, 10 (5) (2000)575-600. “Specific CA inhibitors”, on the other hand, is to beunderstood to mean compounds which largely inhibit only one CA subtype(e.g. CA V) or a defined group of CA subtypes. The use of conventionalCA inhibitors for the specific treatment and/or prophylaxis of obesityis not known.

It is generally accepted that the overwhelming majority of known casesof obesity are attributable to the comparatively excessive proportion ofexogenous fats in the food intake. In highly developed countries likethe USA, up to 30% of the food consumed by obese people is in the formof fats. According to current knowledge, fatty acid deposits leading toobesity accordingly originate predominantly from an excessive intake ofalimentary fats. These cannot be influenced by the inhibition of DNL,which targets carbohydrate metabolism.

Thus, although a reduction in the storage of fatty acids in adipocytesis also achieved by DNL inhibition, the latter has hitherto beenregarded as an unsuitable starting point for the treatment and/orprophylaxis of obesity in man because of its inherently smallcontribution to the storage of fatty acids in body cells. A summary ofthe opinions expressed by experts in this subject can be found forexample in M. K. Hellerstein, European Journal of Clinical Nutrition 53(1) (1999) 53-65. This view prevailing among experts has so far stood inthe way of the specific search for drugs for the treatment and/orprophylaxis of obesity which are based on the principle of DNLinhibition, or of their development.

Within the scope of the present invention, it has now been found,surprisingly, that compounds which are capable of inhibiting DNL inmammals, especially man, can be used effectively for the treatmentand/or prophylaxis of obesity, particularly if these compounds areadministered to the patients in question over prolonged periods of e.g.more than six weeks. Accordingly, over prolonged periods, significantreductions in the body weight of obese persons can be achieved by DNLinhibition even though DNL per se makes only a relatively smallcontribution to the body fat stored in adipocytes. Thus, by means of DNLinhibition over prolonged periods, this inherently small effect canaccumulate in such a way that it makes a significant overallcontribution to the reduction in body weight.

Using the compound topiramate as an example, it can be shown that themethod according to the invention is indeed suitable for specificallydiscovering compounds suitable for the treatment and/or prophylaxis ofobesity. Topiramate is an antiepileptic known from EP 0 138 441 A2.Topiramate is also known to have a multifactorial pharmacologicalspectrum of action. Thus topiramate can block the tension-dependentsodium channels and hence stabilize the membrane potential of cells; itactivates the GABA receptors and thereby enhances GABA-mediatedinhibition; it acts as a carboanhydrase inhibitor, and it is capable ofinhibiting the AMPA/kainate receptors, a subtype of the glutamatereceptors, thereby inhibiting the appearance of AMPA-induced flows.Accordingly, topiramate exhibits pronounced effects on the CNS. It isnot known whether the antiepileptic properties of topiramate areattributable to the pharmacologically relevant factors mentioned above.

It is known from WO 98/00130 that topiramate also possessespharmacological properties which make it appear suitable for thetreatment of obesity. These properties were discovered by chance as sideeffects in long-term studies on epileptic patients. It is not yet knownwhich pharmacological properties of topiramate are responsible for theweight loss observed in epileptic patients. Topiramate is one of a groupof anticonvulsant compounds of a general formula I indicated in WO98/00130 as suitable for the treatment of obesity.

By the method according to the invention, it was now possible to showthat topiramate is a potent CA inhibitor, especially a potent inhibitorof carboanhydrases of subtypes II and V occurring in mammals, and thattopiramate is capable of effectively inhibiting DNL in mammalian cells.Thus the method according to the invention made it possible for thefirst time to prove that the hitherto inexplicable pharmacological sideeffects of topiramate leading to weight loss in epileptic patients arebased substantially on its ability effectively to inhibit DNL inmammals. It is accordingly to be expected that the method according tothe invention will make it possible in the future to discover compoundssuitable for the treatment and/or prophylaxis of obesity. The methodaccording to the invention thus opens up for the first time thepossibility of discovering, with comparative speed and ease,pharmacologically potent compounds which act according to the principleof DNL inhibition. By virtue of the present invention, compounds actingaccording to the above-mentioned principle and suitable for thetreatment and/or prophylaxis of obesity can now be selected, at least ina first preliminary selection process, without lengthy and expensive invivo tests such as test-animal feeding experiments.

These results are surprising as earlier investigations into thepharmacological properties of topiramate indicated that itsCA-inhibitory activity was always unexceptional or even rather small andof no therapeutic importance (cf. e.g. B. E. Maryanoff et al., Journalof Medicinal Chemistry 30 (1987) 880-887; B. E. Maryanoff et al.,Journal of Medicinal Chemistry 41 (1998) 1315-1343; S. J. Dodgson etal., Epilepsia 41 (1) (2000) p. 35-p. 39). In the earlier investigationsmentioned above, the CA-inhibitory activity of topiramate was alwaysdetermined using test solutions containing CA from different mammals,which still contained various body constituents such as blood or organtissue.

In a first embodiment of the method according to the invention,compounds suitable for the treatment and/or prophylaxis of obesity canbe discovered by selecting as suitable those compounds which are capableof inhibiting the activity of at least one carboanhydrase occurring inmammals. For example, at least one test compound can be brought intocontact with at least one carboanhydrase and those compounds whichinhibit the activity of at least one carboanhydrase can then beidentified in a manner known per se. In this embodiment it is preferableto use carboanhydrases which occur in mammals such as man or rodents,for example rats, mice or guinea-pigs, especially carboanhydrases ofsubtypes II and/or V. It is particularly preferable to usecarboanhydrases which occur in man. For example, suitablecarboanhydrases can be isolated from the above-mentioned mammals andpurified if desired, or they can preferably be prepared by chemical orbiotechnological methods known per se.

In a preferred variant of this first embodiment, the change in activityof the carboanhydrases under the influence of the test compounds can bedetermined in an in vitro enzyme activity test known per se, thecarboanhydrases being present as isolated enzymes which have been atleast substantially freed of impurities. It is preferable to usecarboanhydrases which have been prepared by chemical or biotechnologicalmethods as these can be used in especially pure form. In vitro activitytests for determining the activity of carboanhydrases are known per se.The in vitro enzyme activity tests which are suitable within the scopeof the present invention to determine changes in the activity ofcarboanhydrases and include, for example, the measurement of the changein pH value under the influence of carboanhydrases, (cf. K. M. Wilbur,N. G. Andersen, Journal of Biological Chemistry 176 (1948) 147-154; G.Sanyal, T. H. Maren, Journal of Biological Chemistry 256 (1981)608-612), the stop-flow-measurement method (cf. R. G. Khalifah, Journalof Biological Chemistry 246 (1971) 2561-2573) or the4-nitrophenylacetate-esterase method (cf. Y. Pocker, J. T. Stone,Journal of the American Chemical Society 87 (1965) 5497-5498). In thelatter test, the rate of hydrolysis of 4-nitrophenylacetate under theinfluence of the carboanhydrases to be investigated is determined, inwhich the property of carboanhydrases also to act as esterases isutilized.

According to the invention, a test method for determination of theCA-inhibitory properties of compounds which is described by C. T.Supuran et al., European Journal of Medicinal Chemistry 33 (1998)577-594 (cf. p. 592 in particular; cited hereafter as “Supuran et al.”)or by A. Scozzafava et al., Journal of Medicinal Chemistry 42 (1999)3690-3700 (cf. p. 3697 in particular; cited hereafter as “Scozzafava etal.”) is preferentially suitable in this context. Within the scope ofthe disclosure of the present invention, express reference is herebymade to these test methods described by Supuran et al. and Scozzafava etal. Compounds which have IC₅₀ values of at least 10 μmol/liter or below(=higher activity) in one of the above-mentioned in vitro standardactivity tests according to Supuran et al. or Scozzafava et al. can beselected as suitable CA-inhibitory compounds (=CA inhibitors) in termsof the present invention. If the activity of human CA subtypes is to bedetermined, methods other than the 4-nitrophenylacetate-esterase methodmay be better suited. In particular, methods which make it possible alsoto have relatively rapid reactions may be suitable.

In an enzyme activity test (originally described by Y. Pocker and J. T.Stone, Biochemistry 6 (1967) 668-678) operating according to the4-nitrophenylacetate-esterase method described by Scozzafava et al.(vide supra) in terms of the present invention it was demonstrated thattopiramate has a pronounced inhibitory action on human carboanhydrase ofsubtype II (IC₅₀=5 nmol/liter) obtained by biotechnological methods, andthat this inhibitory action is considerably stronger than the inhibitoryaction caused by the conventional CA inhibitors acetazolamide ormethazolamide measured as reference substances. The human carboanhydraseof subtype II was obtained by the method described by Scozzafava et al.

In the same test, it was demonstrated that topiramate also has apronounced inhibitory action on carboanhydrase of the subtype Va of mice(=mCA Va) which is obtained by biotechnological processes (IC₅₀=74nmol/l). In a departure from the test procedure described by Scozzafavaet al, in this case the enzyme mCA Va was used in a concentration of 120nM. The mCA Va was obtained in known manner by the method described byH. R. Heck et al., Journal of Biological Chemistry 269 (1994)24742-24746. To this end, the strain of bacteria Escherichia coli BL 21(DE3) was used, which was used with a plasmid vector which contained thesequence coding for mCA Va under the control of the T7 promoter whichcan be induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Thebacterial culture was inoculated at 37° C. with stirring into aLuria-Bertani liquid medium containing ampicillin (100 μg/ml) and itsgrowth was monitored by spectrophotometry at 600 nm. Once the bacterialculture had entered the exponential growth phase, IPTG was added in afinal concentration of 1 mmol/liter. After 3 hours incubation time (37°C. with stirring), the bacterial culture was centrifuged at 7000×g for15 minutes, and the supernatant was discarded. The resulting pellet wastaken up in 0.1 vol twice-distilled water, and lysozyme (100 μg/ml) wasadded thereto. The cell lysis took place under ultrasound treatment. Tothis end, 10 ml aliquots of the resulting bacterial suspension werepoured into a glass vessel which was open at the top, and each of thesesamples was treated with ultrasound 4 times for 3 minutes. After eachultrasound pulse, the absorption of the samples was determined at 600nm, for which each 100 μl of a-sample was diluted with 900 μltwice-distilled water. The end point was reached once the value of the600 nm absorption of a sample was about 1/10 of the initial value. Oncecell lysis had taken place, CaCl₂-binding buffer (Stratagene) was added,and the resulting cell lysate was poured on to a calmodulin Affinityresin column for purification. The purification is based on the highaffinity of the calmodulin domain which is bound to the resin to thecalmodulin binding peptide tag which is present at the N-terminal end ofthe expressed mCA Va protein. The purification was effected in knownmanner (cf. manual “Affinity LIC Cloning and Protein Purification KitManual” from Stratagene).

In a second embodiment of the method according to the invention,compounds suitable for the treatment and/or prophylaxis of obesity canbe discovered by selecting those compounds which are capable of reducingthe amount of metabolic products formed in the citric acid cycle ofisolated living mammalian cells or of lipidic secondary products of thecitric acid cycle. Suitable metabolic products of the citric acid cyclewhose amount is measurably reduced under the influence of the testcompounds include acid-soluble metabolic products such as citrate,maleate, fumarate and/or α-ketoglutarate. Citrate is preferred.Furthermore, lipidic secondary products of the citric acid cycle, suchas free fatty acids, are suitable as metabolic products. Lastly, in thissecond embodiment, the ability of the test compounds to inhibit theactivity of at least one carboanhydrase occurring in mammals is alsodetermined, although the test model used is set up differently from thefirst embodiment mentioned. The process of the second embodiment isbased on the principle of determining in known manner the uptake ofradioactivity from substrates of the citric acid cycle labeled with the¹⁴C-isotope into metabolic intermediate products or secondary productsof the citric acid cycle of isolated, living mammalian cells andcomparing the results obtained with the results obtained under otherwiseidentical conditions, but under the influence of CA-inhibitorycompounds. Isolated living cells of rodents such as rats, mice orguinea-pigs, or of man, are preferably used in this variant of themethod. Human cells are preferred. If cells of rodents are used, thesecan be adipocytes or hepatocytes. Adipocytes of rodents are preferred.If human cells are used, adipocytes or hepatocytes may be used. Humanhepatocytes are preferred. The mammalian cells used in this variant ofthe method can be obtained by conventional culture and/or cloningprocesses. Natural or biotechnologically modified mammalian cells can beused. If the uptake of radioactivity in acid-soluble intermediateproducts of the citrate cycle is to be determined, preferably¹⁴C-hydrogen carbonate (=NaH[¹⁴C]O₃) is used as the ¹⁴C-labelledsubstrate. If the uptake of radioactivity in lipidic secondary productsof the citrate cycle is to be determined, preferably [U-¹⁴C]glucose isused as the ¹⁴C-labelled substrate.

A preferred variant of this second embodiment for determining theability of compounds to inhibit DNL in mammals is indicated by S. A.Hazen et al. in FASEB Journal 10 (4) (1996) 481-490 (cited hereafter as“Hazen et al.”). Within the scope of the disclosure of the presentinvention, express reference is hereby made to this test methoddescribed by Hazen et al. Compounds which, in this test according toHazen et al., significantly inhibit the incorporation of radioactivityfrom ¹⁴C-labelled substances which can serve as precursors in the citricacid cycle, for example the incorporation of ¹⁴C-bicarbonate intocitrate, maleate, fumarate and/or α-ketoglutarate, with an IC₅₀ value ofat least 10 μmol/l or below (=higher activity) can be selected assuitable compounds in terms of the present invention.

In a test model corresponding to that described by Hazen et al., whichwas carried out within the scope of the present invention, topiramateexhibited a pronounced inhibitory effect on the formation ofacid-soluble metabolic products of the citric acid cycle of ratadipocytes of the 3T3-F442A cell line obtained by biotechnologicalmethods. This inhibitory effect of topiramate (IC₅₀=348 nmol/liter) wasmarkedly more pronounced than the effect of the conventional CAinhibitor ethoxzolamide measured as a reference substance.

In a particularly preferred variant of the method according to theinvention for discovering compounds suitable for the treatment and/orprophylaxis of obesity, those compounds are selected which, in theabove-mentioned first embodiment of the method, especially in anabove-mentioned in vitro enzyme activity test, have been selected assuitable for inhibiting at least one carboanhydrase occurring inmammals, and which additionally, in the above-mentioned secondembodiment of the method, have been selected as suitable for reducingthe amount of metabolic products formed in the citric acid cycle ofisolated living mammalian cells as well. In the first embodiment of themethod, the ability of compounds to inhibit carboanhydrases occurring inmammals can be checked rapidly and effectively. The second embodiment ofthe method provides, inter alia, clues as to whether the compoundsinvestigated are also capable of penetrating mitochondria of livingmammalian cells where CA V is located. Suitable compounds can beselected by carrying out the first and second above-mentionedembodiments in parallel or successively in either order.

According to the invention, compounds which are capable of inhibitingDNL in mammals are suitable for the preparation of drugs for thetreatment and/or prophylaxis of obesity. Compounds selected here arethose which are capable of inhibiting at least one carboanhydraseoccurring in mammals. The selected compounds must of course bephysiologically compatible and meet the demands generally made onpharmaceutical active substances, e.g. regarding safety andcompatibility. It is preferable to use compounds which are capable ofinhibiting carboanhydrases of subtypes II and/or V occurring in mammals.Particularly preferred compounds are those which are capablespecifically of inhibiting CA II and/or CA V, especially CA V.

Previously known compounds with a CA-inhibitory effect which cause areduction in patients' body weight after prolonged administration, forexample topiramate, also have pronounced effects directed towards theCNS, for instance anticonvulsant effects. Side effects directed towardsthe CNS are often undesirable for compounds which are intended forlong-term administration aimed at the treatment and/or prophylaxis ofobesity. Compounds which have been selected by the above-mentionedmethod according to the invention as suitable for the treatment and/orprophylaxis of obesity can therefore additionally be tested for effectsdirected towards the CNS. For example; the compounds can be tested forany anticonvulsant properties which may be present. An example of asuitable method of testing compounds for anticonvulsant properties isthe so-called “supramaximal electroshock test” (=SES test, occasionallyalso referred to as the “maximal electroshock test” or MES test)according to G. Chen et al., Proceedings of the Society for ExperimentalBiology and Medicine 87 (1954) 334-339 (cited hereafter as “Chen etal.”) and J. E. P. Toman et al., Journal of Neurophysiology 9 (1946) 231(cited hereafter as “Toman et al.”). Within the scope of the disclosureof the present invention, express reference is hereby made to this testmethod described by Chen et al. and Toman et al. Thus compounds whichare aimed at the treatment and/or prophylaxis of obesity byadministration to patients over prolonged periods should besubstantially ineffective in the above-mentioned SES test according toChen et al. and Toman et al., and should preferably have no effects tobe regarded as significant according to the criteria conventionallyapplied to this test, even in higher doses of at least 100 mg/kg p.o.(i.e. protective dose, PD₅₀, according to G. Chen et al. ≧100 mg/kgp.o.; the PD₅₀ values given by G. Chen et al. substantially correspondhere to the more common indication of dose as the minimum effectivedose, MED). Compounds which have been selected as suitable in theabove-mentioned method of discovering compounds, and which additionallyare substantially ineffective in the above-mentioned MES test accordingto Chen et al., are particularly suitable for the preparation ofpharmaceutical compositions for the treatment and/or prophylaxis ofobesity. The SES (or MES) tests are standard pharmacological tests andcan be performed as routine methods by appropriate service providers(e.g. “Panlabs”).

The compounds identified by the method according to the invention asbeing suitable for the treatment and/or prophylaxis of obesity canusually be contained as drugs with conventional pharmaceutical auxiliarysubstances in galenic formulations, e.g. tablets, capsules,suppositories or solutions. These galenic formulations can be preparedby methods known per se using conventional solid or liquid excipients,e.g. lactose, starch or talcum or liquid paraffins, and/or usingconventional pharmaceutical auxiliary substances, for example tabletdisintegrants, solubilizers or preservatives. Pharmaceuticalpreparations suitable according to the invention are for example alsoknown from EP 0 138 441 A2 and from WO 98/00130.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof.

1. A method of preparing a pharmaceutical composition for treating orinhibiting obesity, said method comprising: identifying a compound whichinhibits de novo lipogenesis in a mammal, wherein said compound isidentified as a compound which inhibits de novo lipogenesis by measuringinhibitory activity of at least one candidate compound on carboanhydraseactivity of at least one mammalian carboanhydrase, selecting saidcandidate compound as a compound which inhibits de novo lipogenesis ifsaid candidate compound exhibits inhibition of carboanhydrase activityof at least one mammalian carboanhydrase, and incorporating the selectedcompound which inhibits de novo lipogenesis with at least oneconventional solid or liquid excipient or at least one conventionalpharmaceutical auxiliary substance in a pharmaceutical composition fortreating or inhibiting obesity.
 2. The method according to claim 1,wherein said at least one mammalian carboanhydrase comprises a mammaliancarboanhydrase of subtype II or subtype V.